In published studies, we have demonstrated that DAC and DP synergistically induce expression of a variety of C-T genes including NY-ESO-1 and MAGE-A1 in lung cancer cells under conditions that restore expression of tumor suppressors such as p16 and RASSF1A that have been silenced by promoter hypermethylation mechanisms. Notably, this drug treatment regimen is insufficient to mediate induction of these genes in proliferating NHBE cells, possibly due to global methylation status in these cells. Recently, we reported that the germ cell-restricted transcription factor BORIS (brother of the regulator of imprinted sites) is activated during pulmonary carcinogenesis, and that BORIS expression coincides with de-repression of NY-ESO-1, as well as MAGE-A1. Furthermore, we have shown that a CTCF-to-BORIS-shift in occupancy of the NY-ESO-1 and MAGE-A1 promoters coincides with de-repression of these C-T genes in lung cancer cells. More recently, we have observed that BORIS recruits Sp1 to augment C-T gene expression in lung cancer cells. Collectively, these publised data indicate that similar to normal gametogenesis, competition between BORIS and CTCF may be a common mechanism regulating C-T genes during malignant transformation, and that aberrant BORIS expression may contribute to the DNA methylation paradox in lung cancer cells. Ongoing studies have been devoted to further defining the mechanisms by which BORIS mediates C-T and T-S gene expression during malignant transformation. We have identified several lung cancer lines with high level BORIS expression but no MAGE-A1, MAGE-3, or NY-ESO-1 expression. Interestingly, MAGE as well as NY-ESO-1 expression can be induced in these cells following sequential DAC/DP exposure, suggesting that drug treatment either induces expression of a requisite co-factor, or inhibits expression of a repressor of BORIS-mediated activation of C-T genes. Experiments using a variety of techniques including yeast-two hybrid, chromatin immunoprecipitation (ChIP), pyrosequencing, and protein co-IPs are presently underway to identify which additional co-factors are required for BORIS-mediated de-repression of C-T genes, relative to those mediating aberrant silencing of T-S genes in lung cancer cells. In collaboration with Dr. Victor Lobanenkov from NIAID, we have cloned 25 BORIS splice variants, and are presently characterizing expression of these variants in cultured lung cancer cells. We are initiating collaborations with Dr. Lloyd Old (Ludwig Cancer Institute) and Dr. Nasser Altorki (Cornell University Medical Center) in order to comprehensively examine if expression of BORIS splice variants correlates with unique C-T and T-S gene expression profiles in primary lung cancers, and prognosis in patients with these neoplasms. Briefly, these studies will involve blinded analysis of RNA and DNA samples from lung cancer patients with known outcomes utilizing customized oligo and DNA methylation arrays as well quantitative RT-PCR experiments. These studies are expected to yield insight regarding global patterns of epigenetically-regulated gene expression in lung cancer cells, and define the potential clinical relevance of BORIS activation during pulmonary carcinogenesis. In related studies, we have sought to investigate if BORIS can initiate and maintain the malignant phenotype of lung cancer cells. In ongoing experiments, short term cultures of normal human bronchial epithelia (NHBE), small airway epithelial cells (SAEC), human bronchial epithelial cells that have been immortalized by cdk4 and h-tert with or without over-expression of EGFR and k-ras (HBEC), SV40 T-antigen-immortalized bronchial epithelial cells (BEAS), and several lung cancer cell lines, which exhibit different BORIS, C-T, and T-S gene expression profiles have been exposed to NM, DAC, DP, or sequential DAC/DP using our standard treatment regimen. Quantitative RT-PCR, DNA methylation, and long-oligo array experiments are presently underway to examine expression levels of a variety of imprinted, C-T, and T-S genes that are known to be induced/repressed via epigenetic mechanisms in lung cancer cells. Several BORIS splice variants that appear to be preferentially activated in lung cancer cells are being cloned into tet-regulated lentiviral vectors; these vectors will be used to transduce primary as well as immortalized respiratory epithelia to ascertain if BORIS expression is sufficient to induce malignant transformation, as assessed by in-vitro proliferation, soft agar, matrigel, and tumor xenograft assays. The role of BORIS in maintaining the malignant phenotype will be assessed by constitutive over-expression of BORIS in BORIS-deficient lung cancer cells, and knock-down of BORIS in cells exhibiting high level BORIS expression, followed by exposure to a variety of relevant chemotherapeutic agents and ionizing radiation. Collectively, these studies may provide further insight regarding the clinical significance of BORIS activation during pulmonary carcinogenesis. Although important with regard to the development of novel cancer therapies, analysis of epigenetic mechanisms in tumor cells provides limited information pertaining to chromatin remodeling events associated with initiation of malignant transformation. Surprisingly little information is available concerning epigenetic response in respiratory epithelia exposed to tobacco smoke. Recently, we hypothesized that tobacco smoke will induce epigenetic alterations in cultured normal respiratory epithelia that are characteristic of established lung cancers, and that the relative sensitivity of short-term as well as immortalized respiratory epithelial cells to tobacco smoke will reflect the plasticity of the epigenome as evidenced by response to chromatin remodeling agents. As such, we have sought to develop an in-vitro model system for investigating tobacco-mediated epigenetic events in normal respiratory epithelial cells. Briefly, NHBE, SAEC, and HBEC have been exposed to TSC. Micro-array, quantitative RT-PCR, methylation-specific PCR (MSP), pyrosequencing, western blot, and ChIP techniques have been utilized to ascertain if induction/repression of gene expression by TSC coincides with reproducible changes in DNA methylation and the histone code. Our preliminary data suggest that under physiologic exposure conditions, TSC induces epigenetic alterations in normal respiratory epithelia that are characteristic of fully-transformed cells. These provocative findings support further refinement and validation of this innovative model, which could prove considerably useful for elucidating the sequence of epigenetic events associated with tobacco-induced lung cancers. Data from our ongoing laboratory experiments have provided the rationale for a series of clinical protocols evaluating the feasibility of utilizing DNA demethylating agents and HDAC inhibitors for the treatment of thoracic malignancies. During the past several years, approximately 90 patients with thoracic malignancies (predominantly lung cancer) have been treated with a variety of chromatin remodeling agents on protocols conducted in our Section. These trials have been designed to recapitulate in clinical settings drug exposure conditions used in our published laboratory experiments, and have relied extensively on the evaluation of molecular endpoints in pre and post- treatment biopsies using IHC, MSP, quantitative RT-PCR, long-oligo array, and pyrosequencing techniques. Collectively, these trials have established proof of concept pertaining to the use of chromatin remodeling agents for manipulating gene expression in primary thoracic malignancies. Results of the arrays experiments have provided insight into novel mechanisms of drug activity in vivo, enabling more rational development of chromatin remodeling agents for cancer therapy